PROJECT SUMMARY The management of pain?both acute and chronic?can be a frustratingly futile endeavor for both patients and clinicians. Desperate attempts at treatment with opioids and other narcotics has led to a heartbreaking and calamitous epidemic of addiction to prescription painkillers. This epidemic has prompted federal agencies and the pharmaceutical industry to work toward the identification of the next generation of analgesics. Unfortunately, there are few adequate model systems currently in use to enable rapid screening of the analgesic properties of drug candidates. There is an acute need for next-generation neural microphysiological systems that are useful for identifying drug candidates for problems such as pain. Most current microphysiogical models of the nervous system tend toward two categories: organoids and microfluidic/ microelectrode chips. We postulate that the unique complexity and structure of the nervous system demand an integrated approach in order to realize designs of neural microphysiological systems that can begin to account for the basic physiological units that assemble to produce emergent behaviors of the nervous system. We propose to develop a human cell-based model of the afferent pain pathway in the dorsal horn of the spinal cord. Our approach is innovative because it utilizes novel human pluripotent stem cell (hPSC)-derived phenotypes in a model that combines the 3D nature of organoid culture with the structural and organizational specificity of microfabricated systems, all on an integrated, custom 3D microelectrode array. The resulting culture platform will be the only available human model of the dorsal horn afferent circuit. The objectives of the proposal will be met in two phases. In the first, we will establish the feasibility of a physiologically relevant, human, 3D model of the afferent pain pathway that will be useful for evaluation of candidate analgesic drugs. In the second phase, we will then improve the physiological relevance of the system by promoting neural network maturation before then demonstrating the system? utility in modeling adverse effects of opioids and screening a library of compounds to validate the model. Completion of the objective will establish novel protocols for deriving dorsal horn neurons from hPSCs and create the first human microphysiological model of the spinal cord dorsal horn afferent sensory pathway.